Abstract The MAP kinase protein family has a critical role in cellular signaling, with the MAP kinase p381 being linked to events controlling rheumatic disease that includes arthritis. p381 MAP kinase is an important drug discovery target, and drug design efforts have largely focused on small molecule inhibitors of the ATP catalytic site. However, these inhibitors have generally exhibited dose-limiting adverse effects due to similarities between the active site of p381 and other kinases. The active form of p381 is alostericaly regulated, through phosphorylation events provided by a set of auxiliary kinases and phosphatases and through substrate binding. Therefore, characterizing other potential sites within p381, which can bind substrates, inhibitors or allosteric effectors would be of significant interest. The preliminary data in this proposal identifies a lead compound bound in both the active site and the C-lobe pocket of p381. This pocket is formed from an extension to the kinase fold, and occurs in only a small group of kinases, the MAP, CDK and GSK families. Significantly, the preliminary structural and computational analyses suggest that this site within p381 is likely to be suitable for the design of small molecules to bind and potentially modulate the shape and interactions of this kinase in predetermined ways. Thus, the proposed research will build on these studies by defining and comparing specific alosteric binding sites of p381. This proposal will test the hypothesis that MAP kinase activity and function can be specifically modulated through the use of short peptides or small molecules, designed to tightly bind allosteric interaction sites. In Specific Aim 1, key functional interactions of the known 'D-motif' allosteric site, which is bound by certain substrates and regulatory enzymes, will be defined in p381. The goal is to produce effector molecules, modulating enzymatic activity and having a binding affinity for this site greater than natural substrates. In Specific Aim 2, the interactions and potential regulatory functions of small molecule and peptide interactions with the less characterized C-lobe pocket will be defined. Overall, these studies will integrate cutting-edge protein crystallography and small-angle x-ray scattering analyses, with in silico virtual docking methods, peptide array studies and in vitro and in vivo kinase activity assays. The expected results will provide key mechanistic insights into kinase allostery, provide new, targeted molecules regulating enzymatic activity, and reveal new therapeutic strategies for MAP kinase-linked diseases without the active site-linked toxicity. Moreover, the results and concepts developed from these pilot studies on p381 are likely to lead to studies that will test the efficacy of further optimized allosteric regulators, in models of rheumatic disease.